This invention relates to methods for the use of phenylbutyric acid and its pharmaceutically acceptable derivatives to treat alpha-1-antitrypsin deficiency in vertebrate animals. More particularly this invention relates to the treatment and prevention of pathologies resulting in alpha-1-antitrypsin deficiency including liver disease and emphysema. More particularly, this invention relates to the use to phenylbutyric acid and its pharmaceutically acceptable derivatives to increase secretion by the liver of alpha-1-antitrypsin in animals with alpha-1-antitrypsin deficiency caused by the protease inhibitor type Z (PiZ) mutation.
Alpha-1-antitrypsin deficiency is a relatively common genetic disorder that predisposes affected individuals to liver disease and/or pulmonary emphysema. The most common type of alpha-1-antitrypsin deficiency termed protease inhibitor type Z (PiZ), is transmitted as an autosomal recessive trait and affects approximately 1 in 1700 live births in most Northern European and North American populations. The PiZ mutation is a single nucleotide substitution that results in a single amino acid substitution (glutamate 342 to lysine). The replacement of glutamate 342 with a lysine apparently prevents normal folding of the protein. Although not all individuals with the PiZ mutation develop clinical symptoms, it is the most common genetic cause of acute and chronic liver disease in children and the most common genetic diagnosis in children undergoing liver transplantation. The incidence of emphysema or destructive lung injury in this population is not known, but cigarette smoking markedly increases the likelihood of lung injury and accelerates the course of the disease in PiZ individuals.
The major physiological function of alpha-1-antitrypsin is the inhibition of neutrophil elastase, cathepsin G and proteinase 3. The alpha-1-antitrypsin produced in individuals with PiZ alpha-1-antitrypsin deficiency is functionally active, although there may be a decrease in its specific elastase inhibitory capacity. The predominant site of alpha-1-antitrypsin synthesis is the liver, however, it is also synthesized in extrahepatic cell types including macrophages, intestinal epithelial cells and intestinal Paneth cells. In human hepatoma cells alpha-1-antitrypsin is synthesized as a 52 kD precursor that undergoes post translational dolichol phosphate-linked glycosylation at three asparagine residues, and also undergoes tyrosine sulfation. The protein is secreted as a 55 kD native single-chain glycoprotein with a half-time for secretion of 35 to 40 minutes. The half-life in plasma of type M alpha-1-antitrypsin (xcex11-AT) (PiM is the normal allotype) is approximately five days. The half-life of the PiZ mutant protein (xcex11-ATZ) is slightly less, but this difference is insufficient to account for the low plasma levels of alpha-1-antitrypsin in homozygous PiZ individuals.
Studies have provided evidence that the substitution of lysine for glutamate 342 in the PiZ mutant reduces the stability of the protein in monomeric form and increases the likelihood that it will form polymers by the so-called xe2x80x9cloop-sheetxe2x80x9d insertion mechanism. Lomas et al., Nature, 357:605-7, 1992. The presence of polymers of alpha-1-antitrypsin in the endoplasmic reticulum (ER) of individuals homozygous for the PiZ mutation suggest that polymerization may be responsible for the retention of xcex11-ATZ in the ER. Further evidence that polymerization is responsible for retention of xcex11-ATZ in the ER has been provided by studies in which the fate of xcex11-ATZ was determined after introduction of additional mutations. For example, a mutation at amino acid 51, which is remote from the Z mutation and which impedes loop-sheet polymerization, was found to partially correct the intracellular retention of xcex11-ATZ in microinjected Xenopus oocytes. Sidhar et al., J. Biol. Chem., 270:8393-96, 1995.
Secretory glycoproteins ordinarily undergo a series of transient interactions with molecular chaperones in the ER until the folding or assembly process is complete. Once a translocation-competent conformation is achieved, secretory proteins dissociate from molecular chaperones to allow for subsequent transport. If a translocation-competent conformation is not achieved, as might occur with the abnormally folded PiZ alpha-1-antitrypsin molecule, the proteins do not dissociate from their chaperones and thus are retained in the ER until degraded. In individuals with PiZ alpha-1-antitrypsin deficiency, xcex11-ATZ is translocated into the lumen of the ER where it associates with molecular chaperones. But, because of its amino acid substitution, the mutant xcex11-ATZ protein is much less efficient at folding into a translocation-competent shape so that only about 15% of the newly synthesized molecules dissociate from their chaperones and proceed to the Golgi.
The major pathological finding of alpha-1-antitrypsin deficiency is periodic acid-Schiff-positive diastase-resistant globules in the ER of liver cells. As discussed previously, the retention of the PiZ mutant form of alpha-1-antitrypsin in the ER is due to the abnormal folding of the PiZ protein which results in a defect in transport of the protein from the ER to the Golgi. Evidence from studies using transgenic mice suggests that the liver injury seen in alpha-1-antitrypsin deficiency is directly due to the retention of the abnormally folded xcex11-ATZ protein in the ER. Carlson et al., J. Clin. Invest., 83:1183-90, 1988; Dycaico et al., Science, 242:1409-12, 1988. The reason that not all individuals with the PiZ mutation develop liver disease appears to be due to differences in the rate of degradation of xcex11-ATZ within the ER. Studies have indicated that individuals that do not develop liver disease (protected individuals) degrade xcex11-ATZ more rapidly that do individuals who develop liver disease (susceptible individuals). Wu et al., Proc. Natl. Acad. Sci. USA, 91:9014-18, 1994. Thus, conditions or treatments that either increase expression of the PiZ gene or decrease degradation of the mutant protein would be harmful, since they would only serve to increase the accumulation of mutant protein in the ER.
The pathogenesis of lung injury in alpha-1-antitrypsin deficiency is attributable to the marked reduction in available alpha-1-antitrypsin activity. Alpha-1-antitrypsin has been found to constitute greater than 90% of the neutrophil elastase inhibitor activity in pulmonary alveolar lavage fluid. Thus, it appears that the destructive lung disease seen in many individuals with alpha-1-antitrypsin deficiency is due to a perturbation in the net balance between elastase and alpha-1-antitrypsin within the lungs. The uninhibited activity of neutrophil elastase, cathepsin G and proteinase 3, in turn, results in slow destruction of the connective tissue integrity of the lungs. This destruction of connective tissue leads to over distension and a reduction in the retractive force of the lungs which results in decreased expiratory airflow. Smoking exacerbates the problem by causing oxidative inactivation of what alpha-1-antitrypsin is present.
At present, treatment options for individuals with pathologies associated with alpha-1-antitrypsin deficiency are limited. Liver disease associated with alpha-1-antitrypsin deficiency is treated by orthotopic liver transplantation. Perlmutter, Ann. Med. 28:385-94, 1996. The limited supply of livers available for transplantation, the need to maintain transplant patients on anti-rejection drugs, and the cost involved in transplantation surgery, point out the need for alternative treatment methods. Patients with emphysema related to alpha-1-antitrypsin deficiency have been treated with purified plasma alpha-1-antitrypsin administered intravenously or by intratracheal aerosol administration. Lezdey et al., U.S. Pat. No. 5,093,316. The efficacy of this treatment regime, however, has yet to be established. Somatic gene therapy to replace the defective alpha-1-antitrypsin gene has been discussed, but has yet to be successfully used. One potential complication to replacement therapy with either purified alpha-1-antitrypsin protein or the alpha-1-antitrypsin gene is that the individuals treated have high levels of free elastase which, if the treatment is effective, would be expected to generate high levels of elastase-alpha-1-antitrypsin complexes. These complexes, through the serpin-enzyme complex (SEC) receptor, stimulate the synthesis of alpha-1-antitrypsin. In individuals with alpha-1-antitrypsin deficiency, this would lead to an increase in the amount of xcex11-ATZ protein retained in the ER predisposing the patient to liver injury. As used herein, the terms liver disease and liver injury include, but are not limited to, hepatic carcinomas.
What is needed, therefore, is a treatment for alpha-1-antitrypsin deficiency which stimulates secretion of the mutant alpha-1-antitrypsin protein by liver cells without increasing synthesis of the protein. Ideally, this treatment should be easy to administer and have few if any side effects, thus making it suitable for long-term administration.
It has been discovered that derivatives of butyric acid and especially phenyl butyric acid increase the secretion of xcex11-ATZ. For a number of years, phenylbutyric acid (PBA) has been used to treat urea cycle enzyme deficiencies where it functions as an ammonia scavenger. Brusilow, Pediatr. Res., 29:147-50, 1991. Butyric acid derivatives, including PBA, have also been used to treat other conditions. Butyric acid derivatives have been shown to influence cell differentiation in a number of cell types and by a variety of mechanisms. Samid, U.S. Pat. No. 5,635,533; Newmark et al., J. Cell. Biochem. Suppl., 22:247-53, 1995; Kruh, Molec. Cell. Biochem., 42:65-82, 1982. Their effect on cell differentiation has led to the use of butyric acid derivatives as chemotherapeutic agents for cancer treatment and prevention. Samid, U.S. Pat. Nos. 5,852,056, 5,654,333; Carducci et al., Clin. Cancer Res., 2:379-87, 1996; Ram et al., Cancer Res., 54:2923-27, 1994. The ability of butyric acid derivatives to stimulate production of fetal hemoglobin has been used as a treatment for hemoglobin disorders. Samid, U.S. Pat. No. 5,712,307; Collins et al., Blood, 85:43-49, 1995; Perrine et al., Am. J. Ped. Hematology/Oncology, 16:67-71, 1994. Phenylbutyric acid has been used to treat X-linked adrenoleukodystrophy by stimulating production of an alternative peroxisomal membrane protein. Kemp, et al., Nature Medicine, 4:1261-68, 1998. Butyric acid derivatives have also been reported to be useful in increasing the presence of chloride transporters in patients with cystic fibrosis. The exact mechanism by which this is accomplished is unknown. Rubenstein et al., J. Clin. Invest., 100:2457-65, 1997; Zeitlin, J. Clin. Invest., 103:447-52, 1999. There is evidence that phenylbutyric acid stimulates production of the chloride transporter. Rubenstein et al., J. Clin. Invest., 100:2457-65, 1997. It has also been postulated that phenylbutyric acid may also act to provide thermal stabilization to the mutant transporter, thus decreasing degradation within the ER and increasing transport to the cell surface. Zeitlin, J. Clin. Invest., 103:447-52, 1999.
Accordingly, the present invention provides a method for the treatment of alpha-1-antitrypsin deficiency, especially alpha-1-antitrypsin deficiency caused by the protease inhibitor type Z mutation (PiZ). Also provided is a method for the prevention, inhibition and/or treatment of liver disease caused by alpha-1-antitrypsin deficiency, especially alpha-1-antitrypsin deficiency caused by the PiZ mutation. Additionally, the invention provides for a method for the treatment, inhibition and/or prevention of emphysema in animals with alpha-1-antitrypsin deficiency, especially alpha-1-antitrypsin deficiency caused by the PiZ mutation.
One aspect of the invention is to provide a method for the treatment of alpha-1-antitrypsin deficiency in vertebrate animals by administration of an alpha-1-antitrypsin secretion stimulating amount of a compound of formula I: 
wherein R0 is an aryl, phenoxy, substituted aryl or substituted phenoxy; R1, R2, R3, and R4 are independently, H, a lower alkoxy, a lower straight or branched chain alkyl or a halogen; and n is an integer from 0 to 2. The method includes pharmaceutically acceptable salts of formula I, mixtures of various compounds of formula I, and mixtures of pharmaceutically acceptable salts of various compounds of formula I.
Another aspect of the invention is to provide a method for the treatment of liver disease caused by alpha-1-antitrypsin deficiency by administering to a vertebrate animal with alpha-1-antitrypsin deficiency an alpha-1-antitrypsin secretion stimulating amount of a compound of formula I, where R1, R2, R3, R4, and n are as described above. Also included in this aspect are pharmaceutically acceptable salts of formula I, mixtures of formula I compounds, and mixtures of pharmaceutically acceptable salts of formula I compounds.
A further aspect of the invention is to provide a method for the prevention or inhibition of liver disease caused by alpha-1-antitrypsin deficiency by administering to a vertebrate animal with alpha-1-antitrypsin deficiency an alpha-1-antitrypsin secretion stimulating amount of a compound of formula I, where R1, R2, R3, R4, and n are as described above. Also included in this aspect are pharmaceutically acceptable salts of formula I, mixtures of formula I compounds, and mixtures of pharmaceutically acceptable salts of formula I compounds.
A further aspect of the invention is to provide a method for the treatment of emphysema in animals with alpha-1-antitrypsin deficiency by administering to a vertebrate animal with alpha-1-antitrypsin deficiency an alpha-1-antitrypsin secretion stimulating amount of a compound of formula I, where R1, R2 , R3, R4, and n are as described above. Also included in this aspect are pharmaceutically acceptable salts of formula I, mixtures of formula I compounds, and mixtures of pharmaceutically acceptable salts of formula I compounds.
Yet another aspect of the invention is to provide a method for the prevention or inhibition of emphysema in animals with alpha-1-antitrypsin deficiency by administering to a vertebrate animal with alpha-1-antitrypsin deficiency an alpha-1-antitrypsin secretion stimulating amount of a compound of formula I, where R1, R2, R3, R4, and n are as described above. Also included in this aspect are pharmaceutically acceptable salts of formula I, mixtures of formula I compounds, and mixtures of pharmaceutically acceptable salts of formula I compounds.
Still another aspect of the invention is to provide a method for correcting alpha-1-antitrypsin deficiency by detecting the presence of alpha-1-antitrypsin deficiency in an animal, stimulating secretion of alpha-1-antitrypsin by administering an alpha-1-antitrypsin secretion stimulating amount of a compound of formula I, where R1, R2, R3, R4, and n are as described above, and monitoring the alpha-1-antitrypsin levels during and after treatment. Also included in this aspect are pharmaceutically acceptable salts of formula I, mixtures of formula I compounds, and mixtures of pharmaceutically acceptable salts of formula I compounds.
Yet another aspect provides a method for stimulating the secretion of alpha-1-antitrypsin by a cell comprising, contacting a cell containing a protease inhibitor type Z (PiZ) mutation with an alpha-1-antitrypsin secretion stimulating amount of a compound of formula I, where R1, R2, R3, R4, and n are as described above, and monitoring the alpha-1-antitrypsin levels during and after treatment. Also included in this aspect are salts of formula I, mixtures of formula I compounds, and mixtures of salts of formula I compounds.